冷冻后真空抽干膀胱脱细胞基质的生物相容性

Biocompatibility of bladder acellular matrix graft after frozen and lyophilized processing

背景:膀胱脱细胞基质因其制作工序简单,具有良好的生物相容性及细胞黏附性,越来越多的被应用在组织工程生物支架方面。目的:验证冷冻后真空抽干的膀胱脱细胞基质的生物学特性。设计、时间及地点:生物材料相容性实验,于2008-05/11在上海组织工程研究与开发中心实验室及上海第六人民医院实验动物房完成。材料:取2只新西兰大白兔,制备膀胱脱细胞基质。方法:正中切开兔膀胱,仔细剥下膀胱黏膜层,置入三蒸水中浸泡24h。放入含0.1%的聚乙二醇辛基苯基醚及0.15%氨水的脱细胞液中,定期更换脱细胞液,浸泡14d。对照组为直接体积分数为75%的乙醇浸泡保存,实验组为-80℃冷冻24h后真空抽干24h,体积分数为75%的乙醇浸泡保存。主要观察指标:分别通过苏木精-伊红染色、Masson染色、扫描电镜及力学测定研究其理化性质;采用兔膀胱平滑肌细胞作为种子细胞,通过细胞黏附实验、细胞活力测定(MTT法)、皮下埋植实验对2种膀胱脱细胞基质的各种生物学特性进行评价比较。结果:苏木精-伊红染色及Mason染色显示2种膀胱脱细胞基质无细胞成分残留,保持较完整的胶原成分;电镜扫描表面均呈裂隙状结构,以胶原为主,适合细胞黏附;力学检测实验组膀胱脱细胞基质保持了原来膀胱脱细胞基质的力学特征,且具有更好的拉伸率;MTT法测定细胞毒性均为0级;与膀胱平滑肌细胞均有良好的黏附性;兔皮下埋植1个月,均与皮下组织黏附良好,有肌肉黏附及血管增生。结论:经冻干后真空抽干的膀胱脱细胞基质具有良好可操作性及拉伸率,且保持了原有的生物相容性,制作简单,是理想的组织工程生物支架。

BACKGROUND：Bladder acellular matrix graft （BAMG） is frequently used for domains of tissue engineering scaffold due to its great biocompatibility and cell adhesion. OBJECTIVE：To verify the biological characteristics of BAMG after frozen and lyophilized processing. DESIGN,TIME AND SETTING： A biocompatibility experiment was performed at Shanghai Tissue Engineering Research and Development Center and Experimental Animal Department of the Sixth People＇s Hospital of Shanghai between May and November 2008. MATERIALS：Two New Zealand rabbits were used in this study for BAMG preparation. METHODS： After midsection of rabbit bladder, mucous membrane of urinary bladder was isolated and dipped in three-distilled water for 24 hours. Thereafter, the samples were incubated with acellular solution containing 0.1% Triton X-100 and 0.15% aqueous ammonia for 14 days. The culture medium was changed regularly. The samples in the control group were stored in 75% ethanol, while samples in the experimental group were frozen for 24 hours at -80 ℃, vacuum-dried for 24 hours, and stored in 75% ethanol. MAIN OUTCOME MEASURES：Biological characteristics of BAMG were detected using hematoxylin and eosin staining, Masson staining, and scanning electron microscopy; biological characteristics were compared between the two groups using cell adhesion test, MTT assay, and subcutaneously embedding test. RESULTS： Hematoxylin and eosin staining and Masson staining revealed that no residual cells were detected in the BAMG, and collagen was intact. Scanning electron microscopy demonstrated that cells exhibited a slit-shaped structure mainly containing collagen which was beneficial for cell adhesion. Mechanical test revealed that the BAMG after frozen and lyophilized processing not only reserved the mechanical properties of the raw BAMG, but also had a great elongation. MTT assay confirmed that cytotoxicity was grade 0, and BAMG had a good compatibility to smooth muscle cells. After subcutaneously embedding for one month, BAMGs had good adhesions to subcutaneous tissues, and muscular adhesion and vascular proliferation were observed. BAMG after frozen and lyophilized processing reserves original biocompatibility and has great elongation; therefore, it will become a useful and ideal biomaterial for tissue engineering scaffold.